We have studied the patterns of expression of various genes during maturation of normal adult human erythroid precursors using a two-phase liquid culture method. In the first phase, peripheral blood mononuclear cells are cultured for one week in the presence of a combination of growth factors, but not of erythropoietin (EPO). During this phase, early erythroid committed progenitors, burst forming units, proliferate and differentiate into colony forming units. In the second phase, the latter cells, following exposure to EPO, continue to proliferate and mature into hemoglobin-containing orthochromatic normoblasts. Cell samples were taken throughout phase II and expression of globins, transcription factors and cytokine receptors was assayed by RT-PCR and quantitated by phospho-imaging. We have divided Phase II into stages: early (days 0-5), intermediate (days 6-10) and late (days 11-15) and looked at maximum expression of each gene. During early phase II, gamma-globin, CP2 and SP1 were expressed at their highest level. As the cells matured during the intermediate period, the EPO receptor (EPO-R) and transcription factors EKLF, GATA-1 and NF-E2 reached maximum expression. In late phase II, beta-globin increased and reached its maximum level of expression. GATA-2 was undetected in these normal cells throughout phase II; however, GATA-2 expression was observed in uninduced erythroleukemic K562 cells. We also found no expression of the GM-CSF-R in these EPO dependent phase II cells. Expression patterns seen included the reciprocal relationship between the gamma and beta globin genes in which gamma-globin expression is maximum in early phase II and decreases as beta-globin began to increase during the intermediate period. This increase in beta-globin is preceded by a peak in expression of GATA-1, EKLF and EPO-R. We have begun using this type of analysis to examine the role of changes in transcription factor expression with respect to the molecular mechanisms of the HbF-inducing agent hydroxyurea (HU). Cells in Phase II culture are treated with HU at concentrations of 100 mM or 200 mM. Data from our initial experiments show HU treatment results in the prolonged expression of gamma-globin with a corresponding delay in beta-globin expression. Associated with the altered expression of the globin genes are changes in the expression of the transcription factors GATA-1 and EKLF. GATA-1 expression increases after HU treatment while EKLF expression is seen to decrease. These changes result in a shift to the right in the expression curves of both transcription factors. Studies underway will examine changes in the expression pattern of these genes in pathological states of erythropoesis and whether forced over- or under-expression of these genes changes the phenotype of these erythroid cells.